Methods for direct synthesis of compounds having complementary structure to a desired molecular entity and use thereof

ABSTRACT

Compounds which possess a complementary structure to a desired molecule, such as a biomolecule, in particular polymeric or oligomeric compounds, which are useful as in vivo or in vitro diagnostic and therapeutic agents are provided. Also, various methods for producing such compounds are provided. These polymeric or oligomeric compounds are useful in particular as antimicrobial agents, receptor, hormone or enzyme agonists and antagonists.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. Ser. No.08/626,342 filed Apr. 12, 1996 which is incorporated by reference in itsentirety herein. This application claims priority to PCT/SE95/00135, inturn, Application No. 9400450-4, filed Feb. 10, 1994.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention pertains to methods for the directsynthesis of compounds, e.g., polymeric or oligomeric compounds, thatpossess a complementary structure to a desired template molecule, e.g.,a compound having biological activity. The present invention furtherpertains to compounds, e.g., polymers or oligomers produced by suchmethods, and the use thereof, e.g., as therapeutics or diagnostics basedon their complementary structure to a molecule having a known activity.The direct synthesis methods provided herein, which are an extension ofthe technique generally known as “molecular imprinting,” provide apowerful means of producing a compound having a desired activity. Whilethe technique should be applicable for the synthesis of a complementarybinding molecule to any desired compound, the most significantapplication comprises direct drug synthesis. As discussed in detailinfra, the subject invention is particularly useful for direct synthesisof agonists or antagonists for desired molecules, e.g., enzymes,hormones, receptors and other proteins; molecules that affect geneexpression, molecules that affect the binding of biomolecules; e.g.,cells or cell-like moieties to other ligands; and the synthesis ofimproved diagnostic agents.

[0004] 2. Description of the Prior Art

[0005] In traditional drug screening methods, natural products aregenerally isolated, e.g., from plant, animal or microbial extracts andtested for biological activity. These methods generally entail complexpurification and characterization procedures, and the eventualidentification of a natural product having biological activity, e.g., anantimicrobial agent. These natural products are used in their nativeform, or more typically they are improved by the synthesis of syntheticanalogs thereof. These synthetic analogs are then tested for biologicalactivity and the most active compounds become the “drug leads.” Thesecompounds are then used to develop the next generation of syntheticanalogs.

[0006] While these methods have resulted in useful drugs, both naturaland synthetic variants, they are generally very inefficient. Typically,testing must be carried out in animals, or potentially in vitro if thereis a suitable in vitro model to test activity. This is problematic asmany assays, in particular animal testing, require large quantities ofcompound. This is disadvantageous as it limits the number of compoundswhich can be feasibly tested.

[0007] Also, such methods are inherently complex and unpredictable.Often it is difficult to predict and establish the structure/activityrelationship among different compounds tested for activity. This isdifficult to assess, especially if the tested compounds varysignificantly in structure. This makes it difficult to determine theparticular portion of the molecule that is significant for activity.Generally, only by screening large numbers of compounds is this able tobe determined.

[0008] Also, such methods are prone to error. Often compounds that scorepositive in in vitro assays, and even animal models, are inactive inhumans. Conversely, compounds which score negative in vitro may actuallybe active but score negative because of solubility problems which enablean otherwise active compound to cross the cell membrane in vivo.

[0009] Recently, in an effort to obviate some of the problems andinefficiencies of traditional drug screening and synthesis methods,random screening techniques have been developed to identify activecompounds. In such methods, a library, which is simply a collection ofdifferent chemical or biological entities, is screened for one or moreproperties, e.g., binding to a particular ligand. Such librariesinclude, by way of example, compound libraries, peptide libraries,oligosaccharide libraries, and nucleic acid sequence libraries.Typically, the compounds in a particular library possess a relatedstructure, origin and/or function.

[0010] A particular type of library used by many research groupsinvolved in drug design is the “combinatorial library.” This simplyrefers to a library in which the individual members comprise systematicor random combinations of a limited set of basic elements. Randomizationmay be complete or partial. For example, some positions of the testedcompounds may be fixed or varied systematically and others randomlyvaried. Typically, the members of a combinatorial library constituteoligomers or polymers, which vary based on the particular monomers, theconnecting linkages, and/or the length of the oligomer or polymer.Ideally, the members of a combinatorial library are selected such thatthey can be screened for a particular activity or activitiessimultaneously. (See Fenniri, “Recent Advances at the Interface ofMedicinal and Combinatorial Chemistry. Views on Methodologies for theGeneration and Evaluation of Diversity and Application to MolecularRecognition and Catalysis,” Curr. Med. Chem., 3:343-378 (1996), for areview of combinatorial library techniques.).

[0011] One particular type of combinatorial library is the peptidelibrary. These libraries may comprise peptides made by synthetic methodsor by microbial synthesis. In particular, the use of phage or bacteriallibraries wherein a phage particulate or bacterium expresses a desiredpeptide on its surface (by operable linkage of the corresponding DNA toa sequence that encodes a surface protein) are well known. Theselibraries are advantageous because peptides comprise structures thatmimic many biological molecules, i.e., proteins. It is possible bysynthetic or biological techniques to generate a large array ofdifferent peptides of a particular size and sequence, which arethereupon screened for a particular desired property. Microbial surfacedisplay libraries are advantageous in that large numbers of differentpeptides may be obtained in large quantities relatively efficiently.(See G. P. Smith and V. A. Petrenko, “Phage Display,” Chem. Rev.,97:391-410 (1997), for a review on phase display libraries.).

[0012] However, these methods also suffer significant disadvantages. Inparticular, peptides are often costly to synthesize, may be unstable(e.g., in the presence of proteases), and often are unable to crosscellular membranes. Therefore, other molecules, i.e., small organicmolecules, still are preferred drug candidates.

[0013] Such compounds can also be screened by library screening methods.However, small molecules often are not trivial to synthesize inquantities necessary for screening. This disadvantage has somewhat beenalleviated by recent methods which have downsized targets to themolecular level, and the automation of screens which have reduced theamount of compound necessary for assay to small amounts. Theseenhancements have enabled the utilization of combinatorial chemistrylibraries instead of traditional chemical compound libraries.Combinatorial chemistry permits the rapid, relatively inexpensivesynthesis of large numbers of compounds in small quantities suitable forautomated assays directed at molecular targets. Numerous research groupsand companies have reported the design of combinatorial chemistrylibraries which exhibit a significant range of structural diversity.(See, e.g., P. M. Doyle, “Combinatorial Chemistry in the discovery anddevelopment of drugs,” J. Chem. Tech. Biotech., 64(4):317-324 (1995);E.M. Gordon, “Libraries ofnon-polymeric organic molecules,” Curr. Opin.Biotech., 6(6):624-637 (1995)). However, such screening processes stillare often ineffective.

[0014] Thus, based on the foregoing, methods that provide for the directsynthesis of compounds having a desired activity, e.g., a desiredbiological activity would be highly desirable. Moreover, compoundsgenerated by such methods would be extremely desirable because of theirpotential application as drugs and diagnostic agents.

BRIEF SUMMARY OF THE INVENTION

[0015] Toward that end, the present inventors have developed a highlyefficient means of directly synthesizing a compound, in particular apolymer or oligomer having a desired function, typically a biologicalactivity, that enables such compound to be used as a drug, catalyst,competitive affinity ligand inhibitor, competitor, agonist, antagonist,or diagnostic agent. The present inventors have in particular developeda highly efficient means for the direct synthesis of compounds, e.g.,polymers or oligomers, that possess a complementary structure to adesired molecular entity, typically a biomolecule, or portion thereof,e.g., the active site, that are useful, e.g., as agonists or antagonistsof enzymes, hormones, receptors, for regulating gene expression, asantimicrobial or antiviral agents, as reaction catalysts, and in generalfor any activity which relies upon the ability of a compound to bind toanother moiety based on its complementary structure.

OBJECTS OF THE INVENTION

[0016] It is an object of the invention to solve the problems ofprevious indirect drug identification methods.

[0017] It is a specific object of the invention to directly produce acompound that possesses a complementary structure to any desiredmolecular entity or a portion thereof, preferably a biomolecule.

[0018] It is a more specific object of the invention to directly producea compound that possesses a complementary structure to a desiredmolecular entity or a portion thereof, comprising the following:

[0019] (i) selecting a desired molecule, typically a biomolecule, towhich a compound, e.g., a polymer or oligomer, having a complementarystructure is to be obtained;

[0020] (ii) contacting such molecule with one or more monomers,optionally in the presence of one or more crosslinking agents, underconditions that allow for such monomers to associate either covalentlyor non-covalently with specific moieties exposed on the chosen compound;

[0021] (iii) optionally adding one or more crosslinking agents, if notalready present, and polymerizing the monomers which are associatedaround the desired compound to produce a compound, i.e., polymer oroligomer, that possesses a complementary structure to specific moietieswhich are exposed on such compound; and

[0022] (iv) recovering the resultant compound, i.e., polymer oroligomer, that possesses a complementary structure to the desiredmolecule by the removal of the compound from the desired molecule.

[0023] An even more specific object of the invention is to provide acompound that possesses a complementary structure to a desired molecularentity, e.g., a biomolecule such as a microbial or mammalian cell orportion thereof comprising the following steps:

[0024] (i) immobilizing a desired molecule, e.g., a microbial cell to asupport, e.g., a thin layer support such as a silicon wafer;

[0025] (ii) coating (e.g., by spin-coating) onto such support whichincludes the immobilized molecule a polymeric coating that can becrosslinked under controlled conditions, e.g., exposure to irradiation;

[0026] (iii) selectively crosslinking only those portions of thepolymeric coating that coat the immobilized molecule, e.g., by use ofirradiation and a photomask to protect other areas of the polymercoating contained on the support;

[0027] (iv) removal of the non-crosslinked portions of the polymericcoating; and

[0028] (v) removal of the crosslinked polymeric layer from theimmobilized molecule which possesses a complementary structure toexposed residues of such molecule.

[0029] This embodiment of the invention is particularly useful forproducing polymers having a complementary structure to microbial orother cells. The resultant polymeric compounds can be used asantimicrobial agents, anti-tumor agents, etc.

[0030] Still another object of the invention is to provide a method forproducing compounds having a complementary structure to a desiredcompound by the following steps:

[0031] (i) selecting and immobilizing a desired molecule to a support;

[0032] (ii) contacting the resultant immobilized molecule support with asolution comprising one or more monomers, and optionally furthercomprising at least one crosslinking agent, and allowing such monomer toassociate around exposed residues of the immobilized molecule;

[0033] (iii) optionally adding at least one crosslinking agent if notalready provided in step (ii) and polymerizing under conditions thatresult in formation of a molecular coating that possesses acomplementary binding structure to the immobilized compound; and

[0034] (iv) removal of the resultant molecular coating from the support,e.g., by chemical means such as hydrolysis, and cleaving the molecularcoating into discrete segments, that possess a complementary bindingstructure to the immobilized compound.

[0035] Another object of the invention is to provide a method forproducing a compound, e.g., a polymer or oligomer, having acomplementary structure to a desired compound, e.g., a biomolecule, bythe following steps:

[0036] (i) providing a support onto which has been immobilized a fixedfirst layer comprising one or more monomers;

[0037] (ii) providing on top of said first layer a second layercomprising one or more crosslinkable monomers, wherein such monomers arefree to randomly move in the second layer;

[0038] (iii) contacting the second layer with a desired molecule, e.g.,an enzyme, and allowing for the crosslinkable monomers in the secondlayer to associate around specific surface residues of such molecule;

[0039] (iv) optionally adding a crosslinking agent, to produce apolymeric or oligomeric compound that possesses a complementarystructure to exposed residues of such molecule, e.g., residues thatconstitute the active site of an enzyme; and

[0040] (v) recovering the resultant compound, e.g., a polymer oroligomer, that possesses a complementary structure to such molecule bydissociation of the layers and removal of the molecule.

[0041] In yet another embodiment of the invention, a compound having acomplementary structure to a desired molecular entity is producedaccording to the following steps:

[0042] (i) obtaining a desired preformed functionalized polymer, whichmay be linear or lightly crosslinked; and containing said preformedfunctional polymer with a desired molecular entity, which may beimmobilized or in solution, e.g., a biomolecule such as an enzyme, suchthat specific functional groups on the polymer interact with themolecular entity;

[0043] (ii) allowing for the interactions between the functional groupson the preformed polymer and the biomolecule to equilibrate;

[0044] (ii) after equilibration, preserving the resultant complementarystructure on the preformed polymer that results after equilibration byone of the following steps:

[0045] (1) altering the functional groups on the preformed polymer thatinteract least strongly with the molecular entity, e.g., bysite-selective chemical modification;

[0046] (2) altering the functional groups on the polymer that interactmost strongly with the molecular entity by site-selective chemicalmodification; or

[0047] (3) crosslinking the polymer; and

[0048] (iv) thereafter separating the resultant polymer from themolecular entity.

[0049] In this embodiment, one or more crosslinkers may be optionallyutilized, e.g., irreversible crosslinkers. As noted, the molecularentity that functions as the template may be immobilized to a support orcontained in solution.

[0050] Another object of the invention is to provide compounds, e.g.,polymers or oligomeric compounds, that are complementary in structure todesired molecules or portions thereof, in particular the active site(s).These molecules included in particular biomolecules such as enzymes,receptors, hormones, growth factors, cytokines, antibodies, antigens,lectins, biological cells, cell vesicles, nucleic acid sequences,peptides, glycoproteins, carbohydrates, and fragments thereof.

[0051] A more specific object of the invention is to provide compounds,e.g., oligomers or polymers, that are complementary in structure to adesired molecule or portion thereof, e.g., the active site(s) thereof,which may be used, e.g., as agonists and antagonists of enzymes,hormones or receptors; modulators of gene expression, catalysts,therapeutic agents, diagnostic agents, antimicrobial agents, antiviralagents, anti-tumor agents, affinity separation medium, or competitiveaffinity ligands.

[0052] Another specific object of the invention is to use the subjectcompounds that possess a complementary structure to a desired compound,e.g., a biomolecule or fragment thereof, in any method wherein the useof a compound having a structure complementary to that of anothercompound or fragment thereof is desirable. These methods will include byway of example diagnostic methods, prophylactic methods, therapeuticmethods, and catalyzed syntheses methods.

[0053] Still another application is to provide therapeutic, prophylacticor diagnostic compositions which comprise a therapeutically,prophylactically or diagnostically effective amount of a compoundaccording to the invention that possesses a complementary structure to adesired biomolecule or fragment thereof.

BRIEF DESCRIPTION OF THE FIGURES

[0054]FIG. 1 schematically represents imprint formation by non-covalentand covalent approaches.

[0055]FIG. 2 depicts schematically the production of a polymeric oroligomeric compound having a complementary structure to a biomolecule,e.g., an enzyme, receptor or antibody. In this schematic, monomers orother molecules are allowed to align along the surface or active site ofa biomolecule, based on their complementary structure to residues on thebiomolecule. These residues may comprise endogenous functional groupswhich alternatively may be derivatized. After alignment, these monomersare polymerized, optionally in the presence of a crosslinking agent. Thebiomolecule is removed to produce a thin-layer polymeric or oligomericcompound that exhibits a complementary structure to the active site ofthe biomolecule.

[0056] FIGS. 3A-3C schematically represents another means for producingpolymeric compounds that are complementary in structure to desiredmolecular entities. In this method, desired molecules are immobilized toa support, contacted with complementary monomer(s) and crosslinker(s),and polymerization effected, to produce a “segment polymer,” whichsegments are subsequently removed from the support, e.g., by hydrolysisor cleavage.

[0057]FIG. 4 (1-25) shows examples of crosslinkable andnon-crosslinkable monomers which are useful in the present invention.

[0058] FIGS. 5A-5C depicts schematically the production of a compoundhaving a complementary structure to the binding site of a molecule,e.g., a biomolecule sequence or enzyme. In this method (see 5C),monomers in the uppermost layer orient themselves in a complementarymanner around specific exposed residues of the biomolecule, and thesemonomers are “frozen” in this complementary arrangement by crosslinking,and the biomolecule removed to produce a plurality of oligomeric orpolymeric compounds that possess a complementary structure to a portion(e.g., active site) of the selected molecular entity, such as an enzyme(see 5D).

[0059]FIG. 6 (1-5) depicts cleavable monomeric crosslinking agentsuseful in the invention.

[0060]FIG. 7 schematically represents a means for producing polymercoatings that are complementary in structure to large biologicalmoieties, e.g., cells. In this method, selective crosslinking ofspecific areas that surround immobilized biomolecules is effected by theuse of irradiation and a photomask.

[0061]FIG. 8 schematically depicts another embodiment of the invention.

[0062] In this embodiment, a preformed polymer and a desired moleculeare placed in contact, wherein such polymer may be immobilized or insolution. After equilibration, the resultant complementary structure ispreserved by chemical means, e.g. by altering specific functional groupsor by crosslinking.

[0063] In this embodiment, one or more crosslinkers may be optionallyutilized, e.g., irreversible crosslinkers.

[0064]FIG. 9 depicts the specific monomers and crosslinkers used inExample 2 which demonstrate the use of two-dimensional movement in orderto acquire anti-idiotype ligand formation.

[0065]FIG. 10 (1-5) depicts perfluorophenylazide-derivatives (1, 2) anda preassembled scaffold element (3) used in Example 3.

[0066] FIGS. 11A-11C depict schematically the use of molecular scaffoldsto “freeze” a self-assembled complex between ligand providing elementsin their interaction with a binding site.

[0067]FIG. 12 depicts schematically the synthesis of a polymericinhibitor of α-chymotrypsin by direct molding of the polymer on theactive site of the α-chymotrypsin enzyme.

DETAILED DESCRIPTION OF THE INVENTION Relationship of MolecularStructure to Function

[0068] The present invention is based in part on the fact that theactivity of molecules, and in particular biomolecules, is correlated totheir structure, which affects their ability to specifically interactwith other molecules, e.g., receptors, hormones, enzymes, nucleic acidsequences, and microorganisms.

[0069] When the binding partner of a compound such as a protein isknown, it is relatively simple to study the interaction of the compoundand its binding partner, and how such binding interaction affectsbiological activity. Moreover, one can screen compounds for theirability to competitively inhibit the formation of compound-bindingpartner complex or to dissociate such complex. Compounds which inhibitcomplex formation and stability of such complexes are likely to affectthe biological activity of the particular compound, if they can beeffectively delivered to the target site of compound-ligand interaction.

[0070] Generally, it is only specific residues of the compound whichinteract with other moieties, e.g., other biomolecules. These residuesare generally on the surface of the particular compound, e.g., anenzyme, biological cell, receptor, etc.

[0071] Moreover, these residues in turn generally interact with specificresidues which are likewise exposed on the surface of a binding partner,e.g., another biomolecule. In the case of proteins, these residuestypically only comprise relatively small surface portions of themolecule.

[0072] These residue binding interactions which affect biologicalactivity and may result in a reaction proceeding and the formation of acovalent bond are the consequence of the aggregate effects of variousnon-covalent interactions, including the formation of salt bridges,hydrogen bonds, van der Waals forces and other electrostaticinteractions. Also, hydrophobic interactions are important instabilizing the conformation of biomolecules such as proteins, and thusindirectly affect ligand binding, although hydrophobic residues areusually buried and are not part of the binding site.

[0073] Thus, if it were possible to directly produce a compoundcomprising specific residues that specifically interact with suchsurface residues of a desired compound, e.g., a biomolecule, suchcompounds would be highly useful since they will likely affect thebiological activity of the desired compound. Moreover, direct productionwould be further advantageous in that it would eliminate, or at leastsubstantially reduce the need for highly complex and often fruitlessdrug screening methods. Moreover, such direct production wouldpotentially give rise to compounds having enhanced properties inrelation to compounds produced by conventional methods, e.g., enhancedsolubility, stability, activity, affinity and/or avidity relative toligands isolated from conventional sources.

[0074] Molecular Imprinting Technology

[0075] The present invention is based in part on the inventors' previousextensive research and knowledge in the area of molecular imprinting.This technique is reviewed in Biotechnology, Vol. 14, pp. 163-170(February 1996), from which much of this discussion is based.

[0076] The concept of molecular imprinting is depicted in FIG. 1. Themolecule to be imprinted is first allowed to form bonds withpolymerizable entities, which are subsequently crosslinked. Followingextraction of the print molecule, specific recognition sites are left inthe polymer where the spatial arrangement of the polymer networkcorresponds to the imprinted molecule. These procedures make use of ahigh percentage of crosslinker resulting in the formation of rigid andinsoluble macroporous polymers. This template-assisted assembly, leadingto an artificial recognition matrix, is thus performed in a very directway.

[0077] The covalent approach requires a polymerizable derivative of theimprint species that is subsequently incorporated into the polymericmatrix during polymerization. These covalent bonds must be cleavable.The most common types of linkages are either esters ofcarboxylic/boronic acids, ketals or imines (Schiff bases). The necessarysynthetic routes to accomplish such derivatives constrain theversatility of the approach and reduces the number of species that canbe imprinted. After the polymer is formed, the imprint species isextracted by cleavage of these covalent bonds, usually by acidhydrolysis. Rebinding of the imprint species to the matrix is thenachieved by re-establishing the covalent bonds between the printmolecule and the matrix.

[0078] The other, non-covalent approach exclusively uses non-covalentinteractions in the recognition of the imprint species. The greater thevariety of interactions that are available between the imprint speciesand the functional monomers, the better the artificial binding sitebecomes. Typical interaction types that have been exploited are ionicinteractions, hydrogen bonds, π-90 -interactions, and hydrophobicinteractions. Since they are strongly dependent on the polarity of thesolvent, the best imprints are made in organic solvents such aschloroform or toluene. When these normally weak interactions have beenestablished in solution, polymerization is initiated and a porouspolymeric matrix is formed around the imprint species. The formedmacromolecular architecture is thus complementary to the shape andfunction of the imprint species. After polymer formation the imprintmolecule can be almost quantitatively recovered by mild extraction fromthe matrix. Association and dissociation of the original print moleculeto the artificial binder takes place without requiring any covalent bondformation or cleavage. The target molecule simply diffuses in and out ofthe complementary sites.

[0079] Because the limited number of synthetic alternatives forreversible covalent interactions reduces the flexibility of thistechnique, the non-covalent protocol may be more versatile. The use ofnon-covalent interactions allows for the selection of several differentmonomers for simultaneous interaction with the imprint molecule. This inturn leads to a higher degree of selectivity of the imprinting site. Ajudiciously chosen “cocktail” of monomers may be the best way of makingtailor-made artificial binding sites.

[0080] Imprint molecules carrying groups that can bind to metals, e.g.,the imidazole groups of histidine, can be used to coordinatepolymerizable metal chelators. This metal coordination approach has beenrecently evaluated. A combination of covalent and non-covalentapproaches may be advantageous for molecules that seem difficult toimprint. In the case of the steroid cholesterol the single hydroxylgroup was modified to a carbonic ester, allowing its incorporation intothe polymer using the covalent imprinting approach. Subsequent rebindingof cholesterol was performed using only non-covalent forces, aftercleavage of the template. A potential problem with this attractiveprotocol is that the binding site is changed by the chemicalmodification of the site after hydrolysis. Thus, there is the risk ofreducing the site selectivity.

[0081] A large number of substances have been imprinted for variouspractical applications. Four main applications include the use ofmolecularly imprinted polymers: (i) as tailor-made separation materials,(ii) as antibody and receptor binding site mimics in recognition andassay systems, (iii) for catalytic applications as enzyme mimics, and(iv) as recognition elements in biosensors.

[0082] However, the use of such techniques for direct synthesis of drugsand in vivo prophylactic or diagnostic agents has not previously beensuggested. Based on their extensive knowledge and expertise in molecularimprinting, the present inventors conceived the idea that it should bepossible to directly synthesize a compound, e.g., a polymer or oligomer,that possesses a complementary structure to a desired compound, e.g.,biomolecule, or portion thereof, and use the resultant compound inapplications wherein a compound having a complementary structure to abiomolecule would be desirable, e.g. therapeutic applications. Asdiscussed, the ability of most biomolecules to function as therapeuticor diagnostic agents hinges upon its structure, and the interaction ofsuch structure with other molecules. Therefore, the present inventionprovides compounds, e.g., polymers or oligomers, useful as drugs, bothprophylactic and therapeutic agents and in vivo diagnostic agents. Thecompounds produced according to the invention are useful as therapeuticor diagnostic agents based on their ability to specifically interactwith and affect the biological activity of a particular biomolecule thatpossesses a complementary structure to such compound.

[0083] In general, the synthesis of a compound that affects the activityof a particular compound will be effected by a method comprising:

[0084] (i) selecting a molecule, preferably a biomolecule such as anenzyme, the activity of which is desirably to be affected (inhibited orenhanced);

[0085] (ii) contacting such molecule, which may or may not beimmobilized, with one or more monomers that associate with specificresidues of such molecule via covalent or non-covalent interactions;

[0086] (iii) polymerizing the monomers which are associated around suchcompound optionally in the presence of a crosslinking agent, which maybe cleavable, under conditions that result in a molecular network.(“coating”) that is comprised on the surface of such compound, whereinsuch molecular network possesses a complementary structure to theselected compound or specific portion(s) thereof; and

[0087] (iv) removing the molecular network (coating) from the selectedmolecule, and cleaving the molecular coating into smaller moieties, asrequired, to produce a compound that possesses a complementary structureto the selected compound or a portion(s) thereof and which compound issuitable for affecting the activity of such compound, e.g., when used asa therapeutic or in vivo diagnostic.

[0088] Thus, in the present invention, similar to molecular imprinting,polymerizable molecules are permitted to associate by complementarybinding (non-covalent or covalent) to specific groups of a biologicalcompound followed by polymerization. However, an important difference ofthe present invention is that the resultant polymers or oligomers form acoating or image around the biomolecule, which coating or image isremoved therefrom, and discrete entities are derived therefrom, whichmay be used, e.g., as therapeutic or prophylactic agents, i.e., drugs.

[0089] Also, another important difference between the polymers oroligomers that result from the subject invention in relation to theproducts that result from traditional molecular imprinting methods istheir size. In general, the polymeric or oligomeric compounds thatresult from the methods of the present invention will possess amolecular weight that ranges from about 1000 to 200,000, more preferablyfrom about 5,000 to 50,000, and most preferably about 20,000 to 30,000.However, these ranges may dependent upon factors such as the particularmethod utilized to produce such compounds, the particular templatemolecule, and the intended application therefor. Generally, if thepolymer or oligomer is to be utilized as an in vivo therapeutic ordiagnostic, it will possess a molecular weight on the lower end of theabove ranges. In general, polymers according to the invention willcomprise over 100 repeat units and oligomers will comprise less thanabout 100 repeat units. This controls the molecular weight. As notedabove, lower molecular weights are preferably particularly fortherapeutic purposes wherein solubility and viscosity are a significantconcern. The upper limit of the preferred molecular weight range willcorrespond to polymers having about 200-300 repeat units. However, thismay vary dependent upon the particular monomers and the intendedapplication thereof.

[0090] Another difference between the polymers or oligomers that resultfrom the subject invention in relation to conventional molecularimprinting methods is their size. In general, the subject polymers oroligomers will be smaller. The specific size will vary dependent uponthe particular method utilized. Preferably, the polymeric or oligomericcompounds will possess an average chain length ranging from 25 angstromsto 5000 angstroms, more preferably from about 250 to 2500 angstroms, andmost preferably about 500 to 1500. This will vary depending upon theintended application. If the polymeric or oligomeric compounds are to beused therapeutically they will typically be of smaller size, e.g., fromabout 500 to 1000 angstroms, or smaller. Alternatively, the subjectcompounds can be used in vitro, e.g., as affinity separation media orcompetitive affinity ligands.

[0091] As discussed above, the present invention contemplates differentmethods for producing the subject polymeric or oligomeric compounds thatpossess a complementary structure to a desired molecular entity, e.g., abiomolecule. Some of these methods are depicted schematically in FIGS.2, 3, 5, 7, 8, 11 and 12.

[0092] For example, FIG. 2 depicts schematically an embodiment whereinan oligomer or polymer having a complementary structure to the activesite of a molecule, e.g., a biomolecule such as an enzyme, is produced.In this method, monomers or other molecules are permitted to align alongthe surface or active site of a biomolecule, based on theircomplementary structure, to residues on the molecule, e.g., those in theactive site of a biomolecule. These residues may comprise functionalgroups, which alternatively may be derivatized. After alignment, themonomers are polymerized, optionally in the presence of a crosslinkingagent. The biomolecule is then removed to produce a thin-layer polymericor oligomeric compound that possesses a complementary structure to aportion of the selected molecule, e.g., active site of a biomolecule.

[0093]FIG. 3 depicts another preferred means of practicing theinvention. In this method, a desired moiety (“print molecule”) isimmobilized to a support, e.g., a polyacrylamide gel or other supportmaterial. (Other support materials include by way of example silica,polysaccharides, organic polymers, metals, alloys and glass, et seq.).This molecule may be immobilized to the support by covalent ornon-covalent means. After immobilization-the support comprising animmobilized print molecule, e.g., an enzyme, receptor, nucleic acidsequence, or other biomolecule is contacted with a solution containingone or more monomers. The monomers are preferably selected such thatthey are functionally complementary to functional groups comprised onthe immobilized print molecule. For example, if the print moleculecontains positively charged moieties, then negatively selected monomersare preferably selected. Typically, the monomer containing solution willcomprise crosslinkers.

[0094] These monomers are permitted to move and become associated aroundthe immobilized print molecule. Thereafter, polymerization is allowed toproceed. Crosslinking agent is preferably added during polymerization ifnot already present in the monomer solution. The polymerization isconducted under conditions that provide for the associated monomer tomaintain a complementary structure to the immobilized print molecule,e.g., an enzyme.

[0095] Polymerization will result in the formation of “segment” polymersas shown in FIG. 3B. After polymerization, the resultant oligomeric orpolymeric segments are released from the solid support, e.g., byhydrolysis. It is important that the polymer be cleaved into smallermolecules, e.g., oligomers, which are suitable, e.g., as therapeuticagents. This may be accomplished by the use of cleavable crosslinkers.Suitable examples thereof include, but are not limited to, cleavablecrosslinkers such as analogs of bis-acrylamide, such asbis-acrylcystamine, N,N-diallyltartardiamide,N,N-(1,2-dihydroxyethylene)bisacrylamide, orN,N′-bis-(acryloyl)cystamine,Nl-(E)-1-(4-vinylphenyl)methylidene)-4-vinylaniline, allyl disulfide, bis(2-(methacrylgyl, oxyethyl)) disulfide.

[0096] Yet another means of practicing the invention is depicted in FIG.5. This embodiment is particularly suitable for producing polymeric oroligomeric compounds that selectively interact with the active site of abiomolecule.

[0097] In this embodiment of the invention, a fixed polymeric monolayerconstituted of particular monomers is produced. For example, this fixedmonolayer may consist of long-chain alkyl thiols as shown in FIGS. 9, 1.Onto this fixed polymeric monolayer, a second layer is made which willbe constituted of desired monomers, and crosslinkable monomers. Forexample, this second layer may comprise long-chain alcohols having thestructure shown in FIGS. 9, 2 and the crosslinkable, functionalalkenyl-structures shown in FIG. 9, 3-6. Unlike the first layer, themolecules are freely able to move within the second layer in a randommanner.

[0098] A desired biomolecule, e.g., an enzyme, is placed on top of thesecond layer. This results in a directed arrangement of thefunctionalized crosslinkable monomers around specific surface residuesof the biomolecule, e.g., the active site of an enzyme or other protein.For example, the active site of an enzyme such as acetylcholine may beplaced in contact with the second layer resulting in the complexation ofthe functional alkenes with specific residues on the enzyme. Thereafter,polymerization is allowed to proceed by the addition of a crosslinldngagent, such as tetramethyldisiloxane. This results in the formation of acrosslinked polymer that is complementary in structure to the activesite of the biomolecule, e.g., an enzyme such as acetylcholine esterase.

[0099] Thereafter, the two layers on the support are cleaved, and thebiomolecule, e.g., an enzyme is removed resulting in the formation of apolymeric or oligomeric compound that is complementary in structure tothe active site of the biomolecule, e.g., acetylcholine esterase.

[0100] In another preferred embodiment depicted schematically in FIG. 7,a desired- molecule, e.g., a microbial or mammalian cell is immobilizedto a support, e.g., a thin layer support such as a silicon wafer. Afterimmobilization, the support, including the immobilized biologicalmolecule, is then coated with a desired polymer. This may beaccomplished by known methods.

[0101] In this embodiment, a polymer is selected which is crosslinkableunder conditions that enable selected areas of the polymer coating to becrosslinked. For example, this may be effected by the use ofphotocrosslinkable polymers.

[0102] In particular, the regions of the polymer coating which surroundthe immobilized compound are selectively crosslinked. This may beeffected by the use of a photomask to protect specific areas not indirect contact with the immobilized molecule, e.g., a bacterial ormammalian cell, and the use of irradiation to initiate crosslinling ofthe non-protected areas surrounding the molecule, e.g., a particularcell. This results in an imprinted polymer network which surrounds(coats) the immobilized biological moiety. This imprinted polymercoating is then removed from the solid support. This polymeric coatingwill possess a complementary structure to exposed residues of theimmobilized moiety, e.g., a cell. This polymeric material therefore maybe used to affect the activity of the immobilized moiety, e.g., amicrobial or mammalian cells. For example, it should be useful as anantimicrobial agent, which should inhibit such cells from infectingsusceptible cells. Also, it should be useful as a cell separation agent.

[0103] This polymeric material also may be attached to other materials,e.g., therapeutic and/or diagnostic agents in order to target suchmaterials to desired cells, e.g., mammalian tumor cells or the site ofinfection.

[0104] This embodiment of the invention, because it requires thecrosslinking of specific areas, e.g., by the use of a photomask, is notpractical for small molecules, such as active sites. Rather, it is bestsuited for larger biological moieties such as microbial and mammaliancells as well as biological surfaces, e.g., tissues.

[0105] In yet another embodiment of the invention depicted schematicallyin FIG. 8, a molecular entity is allowed to interact with a preformedfunctional polymer which can either be linear or lightly crosslinked.The pre-formed functional polymer can interact with the molecular entityvia covalent and/or non-covalent bonds (FIG. 8). After equilibrium,either (a) those functional groups on the polymer which interact leaststrongly with the molecular entity are chemically altered by, forexample, site-selective chemical modification, or (b) those functionalgroups on the polymer which interact most strongly with the molecularentity are chemically altered by, for example, site-selective chemicalmodification, or (c) crosslinking of the polymer can be preformed.Thereafter the polymer is separated from the molecular entity whichacted as a molecular template and purified via an appropriate procedure.In this embodiment the molecular entity used as a template may existfreely in solution or alternatively be immobilized onto a suitablesupport and, optionally, one or more crosslinkers may be used, one ormore of which may be reversible crosslinkers.

[0106] In still another embodiment of the invention, depictedschematically in FIG. 11, a complementary structure to a desiredmolecule, e.g., an enzyme, is produced using molecular scaffolds thatfunction to “freeze” a self-assembled complex between ligand buildingelements in their interaction with a portion of a template molecule,e.g., active (binding) site of an enzyme. In this embodiment, a desiredmolecule, e.g., an enzyme such as crosslinked trypsin, is mixed togetherwith derivatized ligand building elements, e.g., theperfluorophenylazide-derivatives shown in FIG. 10, which are judiciouslyselected such that they fit within the active site of such molecule.

[0107] Thereafter, these ligand elements including the scaffold-bearingelement, are induced to specifically (rather than randomly) interactwith the molecule by effecting such interaction in a suitable solventsystem. For example, in order to reduce non-specific hydrophobicinteractions, the process can be effected in a polar solvent such asacetonitrile. After these specific interactions have occurred, theself-assembled complex between the ligand building elements and theactive site is “frozen,” e.g., by exposure to UV radiation which resultin the azido-functionalities becoming inserted into theamino-functionalities of the scaffold. (See 11C). Thereafter, theresultant active site-binding conditions are separated from the randombinding adducts, e.g., by affinity chromatography.

[0108] In still another embodiment of the invention, depictedschematically in FIG. 12, the invention provides for the directsynthesis of a compound complementary to the active site of a compound,e.g., an enzyme, by directly molding a polymer onto the active site ofsaid enzyme. In this embodiment, an anchoring monomer is allowed tointeract with the molecule, e.g., an enzyme such as α-chymotrypsin, aswell as a filling monomer (that fills active site thereof) such asmethacrylamide or methacrylic acid. Polymerization is then initiated,e.g., by UV irradiation. The resultant polymeric or oligomeric compoundsthat surround the compound are then separated from the compound, e.g.,an enzyme, by hydrolysis. Thereafter, the desired polymeric oroligomeric compounds are isolated, e.g., by affinity chromatography. Forexample, affinity chromatography can be effected using a support thatspecifically interacts with the anchoring monomer, e.g., a His sepharosesupport. The non-bound portion is discarded, and a second affinitychromatography can be effected using an affinity support containing thetemplate molecule, e.g., α-antitrypsin. The resultant polymeric oroligomeric compounds are then eluted from the support and tested foractivity, e.g., inhibiting activity against α-chymotrypsin using astandard activity assay using BTEE.

[0109] As discussed, the subject invention provides compounds, i.e.,polymers or oligomers, that exhibit a complementary structure to desiredmolecules, e.g., biomolecules, or portions thereof, e.g., the activesite. These compounds are useful as in vivo or in vitro therapeutic ordiagnostic agents based on their ability to affect the activity of aparticular biomolecule, e.g., a protein, DNA, virus, receptor, hormone,enzyme glycoprotein, microbial cell, mammalian cell, etc. Also, thesecompounds may be used as competitive affinity ligand inhibitors,competitors, agonists, catalysts, or antagonists. These uses are meantto be exemplary and not exhaustive of the applications of the compoundswhich result from the present invention. Essentially, the subjectcompounds can be used for any purpose wherein a compound. having acomplementary structure to another compound is useful.

[0110] I. Selection or Functional Monomers for Production of SubjectOligomeric or Polymeric Compounds

[0111] The polymerization reaction mixture for the preparation of thesubject complementary compounds usually consists of a desired molecule,e.g., a biomolecule, polymerizable functional monomers, an effectiveamount of one or more crosslinking agents which enable formation of asufficiently rigid polymeric or oligomeric structure, inert solvent, anda free radical or other polymerization initiator if necessary toinitiate polymerization. Mixtures of monomers and crosslinking agentscan be used in the polymerization method.

[0112] Two approaches to the production of a molecular imprint polymerhave been developed, and either can be used in the methods disclosedherein. In the first method, a biomolecule is covalently bound to apolymerizable monomer, and after polymerization, the covalent bond iscleaved to release the biomolecule from the polymeric coating. Usingthis method, a selected biomolecule is attached to a polymerizablemoiety using any appropriate method. The polymerizable biomoleculeshould contain a linkage that can be broken to release the biomoleculeafter the polymeric compound is formed, without adversely affecting thecomplementary structure thereof. The resultant polymer compound shall becleavable into discrete entities suitable for in vivo use.

[0113] In the second method, polymerizable monomers arrange themselvesabout a biomolecule based on non-covalent interactions (such as ionic,hydrophobic, steric, electrostatic, and hydrogen bonding interactions),and after polymerization, the non-covalently bound biomolecule is simplyleached out.

[0114] Any suitable combination of functional monomers, crosslinkers andinitiators that provide an accurate imprint of the biomolecule onpolymerization (a polymer compound confirming a complementary structure)is suitable for use in the present invention.

[0115] In general, the imprinted compound should exhibit as closely aspossible the reverse topology of the biomolecule. For example, if thebiomolecule has an anionic group at a specific location that isimportant to the desired biological activity of the mimic, the imprintedpolymeric compound should have a cationic group at that location. If thebiomolecule has a cationic group at a specific location that isimportant to the desired biological activity of the biomolecule, thepolymeric compound imprint should have a anionic group at that location.

[0116] Preferred classes of monomers and specific monomers include, butare not limited to, the following classes and derivatives thereof:acrylic acid and derivatives (e.g., 2-bromoacrylic acid, acryloylchloride, N-acryloyl tyrosine, N-acryoyl pyrrolidinone), acrylates(e.g., alkyl acrylates, allyl acrylates, hydroxypropyl acrylate),methacrylic acid and derivatives (e.g., itaconic acid,2-(trifluoromethyl) propenoic acid), methacrylates (e.g., methylmethacrylate, hydroxyethyl methacrylate, 3-sulfopropyl methacrylatesodium salt), styrenes (e.g., (2, 3 and 4)-aminostyrene,styrene-4-sulfonic acid, 3-nitrostyrene), vinyls (e.g., vinylchloroformate, 4-vinylbenzoic acid, 4-vinylbenzaldehyde, vinylimidazole, 4-vinylphenol, 4-vinylamine, acrolein), vinylpyridines (e.g.,(2, 3, and 4)-vinylpyridine, 3-butene 1,2-diol), boronic acids (e.g.,4-vinylboronic acid), sulfonic acids (e.g., 4-vinylsulfonic acid), metalchelators (e.g., styrene iminodiacetic acid), acrylamides andderivatives (e.g., N-methyl acrylamide), methacrylamides and derivatives(e.g., N,N-dimethyl acrylamide, N-(3-aminopropyl)methacrylamide),alkenes (e.g., 4-pentenoic acid, 3-chloro-1-phenyl-1-propene)(meth)acrylic acid anhydride and derivatives (e.g., methacrylicanhydride), silicon-containing monomers (e.g.,(3-methacryloxypropyl)trimethoxy silane, tetramethyldisiloxane),polyenes (e.g., isoprene,3-hydroxy-3,7,11-trimethyl-1,6,10-dodecatriene), azides (e.g.,4-azido-2,3,5,6-tetrafluorobenzoic acid), thiols (e.g., allylmercaptan). Acrylate terminated or otherwise unsaturated urethanes,carbonates and epoxies can also be used in this present invention, ascan silicon-based monomers.

[0117] If utilized, the crosslinking agent or agents will preferably beone or several polymeric or oligomeric compound, or a compound thatprovides for cleavage under specific conditions.

[0118] Crosslinking agents that lend rigidity to the subject polymericcompounds are known to those skilled in the art, and include, but arenot limited to, di-, tri-, tetra- and penta-functional acrylates,methacrylates, acrylamides, vinyls, allyls, and styrenes. Examples ofreversible, cleavable crosslinkers which are useful in this inventioninclude, but are not limited by, N,N′-bis-(acryloyl)cystamine,N,N-diallyltartardiamide, N,N-(1,2-dihydroxyethylene) bisacrylamide,N1-((E)-1-(4-vinylphenyl)methylidene)-4-vinylaniline, allyl disulfide,and bis(2-methacryloyloxyethyl))disulfide.

[0119] Any ratio of simple monomers to crosslinking monomers can be usedthat provides a polymeric structure of appropriate integrity, e.g., thatcan be used in vivo. Those skilled in the art can select suitable ratiosof monomers to provide the desired structural integrity.

[0120] In the case of polymeric or oligomeric compounds that are to beutilized in vivo as therapeutics or diagnostics, it is important toselect monomers that are non-toxic. and which exhibit suitable in vivostability and solubility. Preferred examples include, but are notlimited to, acrylamides and acrylates. Alternatively, the polymer may betreated post-polymerization to enhance solubility, e.g., by reactionwith suitable organic molecules.

[0121] Different polymerization methods may be used including freeradical, cationic, and anionic polymerization. Polymerization conditionsshould be selected that do not adversely affect the active conformationof the compound for which a complementary polymeric compound is to beproduced.

[0122] Preferred monomers useful in the invention are reversiblycrosslinking monomer containing Schiffs base linkages. These compoundsare depicted below:

Crosslinkers Containing Schifffs Base-linkages

[0123] Another possibility is the use of disulfide containing analogs ofbis-acrylamide e.g., bis-acrylylcystamine, which can be dissolved with2-mercaptoethanol

[0124] Other useful cleavable monomer crosslinkers include, but are notlimited to, N,N′-di-allyltartardiamide andNN′-(1,2-dihydroxyethylene)bis-acrylamide.

[0125] The subject compounds, dependent upon the particular moleculewith which they are complementary, may be used by way of example asantagonists or agonists of hormones, receptors or enzymes; as modulatorsof gene expression, as antimicrobial compounds, as vaccines, asanti-tumor agents and as wound healing agents.

[0126] A preferred embodiment of the invention involves the productionof compounds having a complementary structure to insulin. The resultantcompounds will be advantageous to native insulin because they should beintrinsically more stable, i.e., more heat, enzymatically, and pHstable. Moreover, these compounds, given their pH stability, should beorally administrable. This is clearly a significant advantage as it mayprovide for the treatment of diabetes without the need for insulininjection.

[0127] Use of the subject compounds as in vivo therapeutic orprophylactic agents or in vivo diagnostic agents will generally entailthe addition of a pharmaceutically acceptable carrier or excipient,e.g., water, phosphate buffered saline, surfactants, adjuvants, etc.Suitable carriers and excipients are well known to those skilled in theart.

[0128] The amount of the particular compound will depend upon factorsincluding its activity, solubility, in vivo stability, and specifictherapeutic or diagnostic application.

[0129] The subject compounds can be administered by any known means ofadministration, e.g., orally, intranasally, intravenously,intradermally, topically, subcutaneously, submuscularly, testicularly,rectally. Preferred means of administration include oral and intravenousinjection.

[0130] A suitable dosage of the subject polymeric drug will generallyrange from about 0. 00001 to 5.00 mg/kg of body weight, more preferablyfrom about 0. 01 to 1.00 mg/kg of body weight.

EXAMPLES Example 1

[0131] This example describes the formation of amolecularly imprintedmaterial using two differently reacting crosslinking monomers, A, and B.By virtue of choosing the mutual reactivity ratios (r) so that theproduct r_(A)r_(B)<1, these monomers will preferably form stretches ofhomopolymers, rather than random, or alternating, copolymers.Polymerization of a mixture of these. crosslinkers will lead to segmentpolymer formation: -A-A-A-A-B-B-B-B-A-A-A-A.

[0132] A solution comprising the two different crosslinkers, ethyleneglycol dimethacrylate (EDMA) (FIG. 4, 23) andN1-((E)-1-(4-vinylphenyl)-methylidene)-4-vinylaniline (VMVA) (FIG. 6,2), together with the functional monomer methacrylic acid (MAA) (FIG. 4,3), in acetonitrile is spraycoated onto the print molecule, immobilizedonto a silicon wafer support (FIG. 3A). Upon exposure to UV-irradiationat 366 nm, polymerization takes place, during which a continuousthree-dimensional segment polymer is formed around the print molecule(FIG. 3B). The thickness of this polymer can be controlled by thespraycoating process, and is normally in the range of 100 nm to 1 μm.

[0133] Treatment of the polymer with acidic/basic water solution for 24hours leads to hydrolysis of the imine-bond of VMVA (FIG. 3C). Thisresults in the dissolution of the VMVA-segments in the polymer, thusleading to the liberation of discrete polymer segment particlesconsisting mainly of EDMA and MAA. The size of the particles can becontrolled by changing the ratio between EDMA and VMVA. In the idealcase, the particles are prepared in the nanometer range. Followingextensive washing and removal of the polymer from the wafer support,these particles can be utilized in rebinding of the print molecule.

Example 2

[0134] This example demonstrates the use of two-dimensional movement inorder to acquire anti-idiotype ligand formation.

[0135] A self-assembled monolayer (SAM), consisting of long-chain alkylthiols (FIG. 9, 1) is built on a gold surface (FIG. 5A). On top of thislayer, a second layer is built, consisting of long-chain alcohols (FIG.9, 2) as well as crosslinkable, and functional alkenyl-structures (FIG.9, 3-6)(FIG. 5B). In the second layer, the molecules are free to movewithin the layer in a random manner. Addition of a solution containingthe target molecule, e.g., acetylcholine esterase (AChE), on top of thesecond layer, results in a directed arrangement of the functionalalkenyl-molecules towards the enzyme. Patches of complexes between thefunctional alkenes and the enzyme takes place on the surface (FIG. 5C).These complexes are subsequently “frozen” by the addition of acrosslinker, such as tetramethyldisiloxane (TMDS)(FIGS. 9, 7)(FIG. 5D).After breakage of the layers, and removal of the enzyme, structuresremain that are complementary to the active site of AChE (FIG. 5E).These polymer compounds may be used to affect acetylcholine esterateactivity. Thus, these compounds, when combined with a pharmaceuticallyacceptable carrier or excipient are useful for treating conditionswherein modulation of acetylcholine esterase activity is therapeuticallydesirable.

Example 3

[0136] This example represents the use of molecular scaffolds to“freeze” a self-assembled complex between ligand building elements intheir interaction with a binding site.

[0137] Crosslinked trypsin (from Altus), a proteolytic enzyme specificfor the cleavage of peptide bonds (-X-Y-) where X can be any amino acidresidue and Y is a positively charged residue, is mixed together withligand building elements labeled with photoactive groups, e.g.,perfluorophenylazido groups (FIG. 10, 1-2), chosen so as to be able tofit into the active site of the enzyme, and a preassembledscaffold-ligand element (FIG. 10, 3) (FIG. 11A). The ligand elements,including the scaffold-bearing element, are prone to interact with theenzyme randomly. In order to enhance non-covalent interactions betweenthe ligand building elements and the enzyme, and to reduce the amount ofnon-specific hydrophobic interactions, the process is performed inacetonitrile. After a period of time when self-assembly is allowed to beestablished (FIG. 11B), the solution is exposed to UV-radiation (254 nm)during which process the azido-functionalities will insert into theamino-functionalities of the scaffold. Finally, the active site-bindingcandidates are separated from the randomly binding adducts via affinitychromatography on a trypsin-column (FIG. 11C).

Example 4

[0138] This example describes a general method for the preparation oftemplated, linear, soluble polymers which display structuralcomplementarity to the original molecule. (Serine protease.)

[0139] The serine protase trypsin (5 mg) is dissolved in aqueous sodiumphosphate buffer (1 mL, pH 7, 0.05M). Alternatively, trypsin can beimmobilized on an inert, insoluble support and then suspended in theaqueous sodium phosphate buffer. Acryloyl 4-aminobenzamidine (5 mg)(acryloyl 4-aminobenzamidine binds strongly to Asp 189 in the activesite of trypsin), acrylamide (70 mg) and TEMED (6 μL) are added;additional functional monomers can also be introduced at this stage ifdesired. The mixture is equilibrated at room temperature for thirtyminutes, degassed by purging with oxygen-free nitrogen and thepolymerization initiated by the addition of 10% w/v ammonium persulphatein water (120 μL). Once the polymerization is complete (2-3 hours) thepolymer is separated from the trypsin and unreacted monomers/initiator.This is achieved via a simple filtration step in the case of immobilizedtrypsin, or by passage of the reaction products through an affinityseparation column which is specific for trypsin in the case ofnon-immobilized trypsin. Thereafter the linear, soluble, templatedpolymeric products are purified via repeated precipitation from waterinto methanol, and characterized via NMR and FTIR spectroscopy. Theinhibitory properties of the resultant purified linear, solublepolymeric compounds versus trypsin are then tested in a standard enzymeassay.

Example 5

[0140] This example describes a general method for the preparation oftemplated, linear, soluble polymers which display structuralcomplementarity to a print molecule, and which are obtained via thesolubilization of insoluble, imprinted cross-linked polymers containingreversible, cleavable cross-linking moieties.

[0141] The serine protease trypsin (5 mg) is dissolved in aqueous sodiumphosphate buffer (1 mL, pH 7, 0.05M). N-Acryloyl 4-aminobenzamidine (5mg) (acryloyl 4-aminobenzamidine binds strongly to Asp 189 in the activesite of trypsin), acrylamide (70 mg), N,N′-diallytartardiamide (5 mg)and TEMED (6 μL) are added; additional functional monomers can also beintroduced at this stage if desired. The mixture is equilibrated at roomtemperature, degassed by purging with oxygen-free nitrogen and thepolymerization initiated by the addition of 10% w/v ammonium persulphatein water (120 μL). Once the polymerization is complete (2-3 hours), thetrypsin and unreacted monomers/initiator are removed by washing from thecross-linked polymer network using 10% v/v acetic acid in watercontaining 10% w/v SDS (5×10 mL), and the polymer then washed thoroughlywith distilled water (5×10 mL) to remove traces of SDS and acetic acid.The cross-linked gel is then treated overnight with 2% w/v aqueousperiodic acid (5 mL), the linear soluble, templated polymer isolated andpurified via repeated precipitation using methanol. The polymer ischaracterized via NMR and FTIR spectroscopy and the inhibitoryproperties thereof versus trypsin tested via a standard enzyme assay.

Example 6

[0142] This example provides a lithographic method for the preparationof templated polymers which are complementary to the print molecules(e.g. proteins and cells) in terms of their surface relief and/orchemical structure (FIG. 7).

[0143] A silicon wafer is aminopropylated and insulin immobilized on thesurface via a literature method (Biochemistry, Vol. 11:2291 (1972)). Asolution of poly(methacrylic acid-co-glycidyl methacrylate) and thephotoacid catalyst generatorp-nitrobenzyl-9,10-dimethoxyanthracene-2-sulphonate in 2-methoxyethanolis then spin-coated onto the surface to the desired thickness (typically1 μm), together with additional functional monomers and/or pre-polymersif desired. A photomask is put in place and the film exposed to filteredEM radiation from a super high-pressure mercury lamp (365 nm) for a setperiod of time (see J. Appl. Polym. Sci., Vol. 50:243 (1993)). Followingdevelopment of the film via treatment with aqueous tetramethylammoniumhydroxide (2% w/v) followed by a post-exposure bake at elevatedtemperature (80° C.), the discrete imprinted particles are released fromthe silicon surface.

Example 7

[0144] This example provides a means for preparing linear, templatedpolymers which bind specifically to integrins, which are cell-surfacebased proteins that are involved in cell-cell or cell-matrixinteractions in biological processes.

[0145] The integrin α_(lib)β₃ (1 mg), N-acryloyl L-arginine (5 mg),N-acryloyl L-glutamic acid (5 mg), acrylamide (50 mg) and TENED (5 μL)are suspended in aqueous sodium phosphate buffer (1 mL, pH 7). Themixture is equilibrated at room temperature, degassed by purging withoxygen-free nitrogen and polymerization initiated by the addition of 10%w/v ammonium persulphate in water (100 μL). After four hours thetemplated polymer is released from the integrin via the addition of thecompeting synthetic tripeptide RGD (100 mg) and the polymer isolated viafractional precipitation with saturated ammonium sulphate. The polymeris then purified via repeated precipitation from water into methanol.

Example 8

[0146] This method describes the synthesis of a polymeric inhibitor ofα-chymotrypsin by direct molding of the polymer on the active site ofsuch enzyme (FIG. 12).

[0147] Bovine α-Chymotrypsin can interact with iminodiacetic acid-Cu(II)complexes via its His-40 (Berna et al., Biochemistry, Vol. 36:6896(1997)). Allyl-2-hydroxy-3(N,N-dicaboxymethylamino)propylether-Cu(II)(Baek, Haupt, Colin and Vijayalakshmi, Electrophoresis, Vol. 17:489(1996)) is used as the anchoring monomer. Methacrylamide and methacrylicacid are used as the filling monomers.

[0148] Crosslinked crystalline α-Chymotrypsin (Altus) (240 mg) is mixedwith 1 ml of a heptane solution containing allyl-2-hydroxy-3(N,N-dicarboxymethylamino)propylether Cu(II) (12.4 mg), methacrylamide (17mg), methacrylic acid (17 mg) and 2,2′-azo-bis-isobutyronitrile (17.5mg) in a 1.5 ml polypropylene test tube. After filling the head space ofthe tube with argon, the tube is cooled on ice. Polymerization isinitiated by UV irradiation at 366 nm for 30 min. The suspension isfiltered and the chymotrypsin crystals are washed four times with 1 Macetic acid in heptane. The filtrates are combined and the solvent isevaporated under vacuum. The remaining polymer is redissolved in 25 mMMOPS buffer pH 7 containing 0.3 M NaCl, and the solution is centrifuged.The supernatant is purified by affinity chromatography on a columncontaining His-Sepharose (Sigma), pre-equilibrated with 25 mM MOPSbuffer pH 7 containing 0.3 M NaCl. The non-retained fraction. isdiscarded, and elution is performed with 0.1 M sodium acetate buffer pH4. The eluted polymer fraction is dialyzed overnight against 50 mMsodium phosphate buffer pH 7. The resulting polymer fraction containingmetal chelate groups can be further fractionated by affinitychromatography on α-chymotrypsin immobilized onto agarose beads (Sigma).The retained polymer molecules are eluted with 50 mM sodium acetatebuffer pH 4, containing 1 M NaCl. The eluted fraction is dialyzedagainst ultrapure water and freeze-dried. The polymer fractions aretested for their inhibitory effect versus α-chymotrypsin in a standardactivity assay using BTEE.

Example 9

[0149] This example describes the synthesis of a polymeric competitorfor antibody binding to its antigen by direct moulding of the polymer onthe antibody's binding site.

[0150] Antibodies are immobilized onto porous silica beads.Polymerization takes place only in the pores of the beads which aresuspended in a perfluorocarbon solution to prevent the polymerizationmixture from exiting the pores. Monoclonal antibodies against lysozymeare used as model system and the resulting polymer specifically inhibitsantigen binding to the antibody.

[0151] 2 g of aminopropyl silica beads (10 μm diameter, average porediameter of 100 Å) is suspended in 5 ml of a solution of 1 M succinicanhydride in THF. The suspension is sonicated for 30 min andsubsequently incubated on a overhead shaker for 5 h at room temperature.The beads are removed by centrifugation and washed by incubation for 1 heach time with THF (2 times) and methanol (3 times) followed bycentrifugation. The solvent is removed under vacuum. The beads are thensuspended in 5 nil of a solution of 0.2 M EDC, 0.2 M NHS and 0.2 Mmethylmorpholine in THF and allowed to react overnight at roomtemperature on a overhead shaker. The beads are removed bycentrifugation and washed by incubation for 1 h each time with THF (2times) and methanol (3 times) followed by centrifugation. The solvent isremoved under vacuum.

[0152] The beads are packed in a FPLC column (10×0.5 cm) and washed with50 mM phosphate buffer pH 7.5, 0.02% Tween-20, for 2 h at a flow rate of1 ml/min. All following steps are carried out at 4° C. 5 ml of asolution of a sheep monoclonal antibody (IgG) against lysozyme (1 mg/mlin 50 mM phosphate buffer pH 7.5, 0.02% Tween-20) is repeatedly (3×)injected into the column at a flow rate of 0.2 ml/min. The column isthen washed with phosphate buffer pH 7.5, for 2 h at a flow rate of 0.5ml/min. The column is wrapped in aluminum foil and a solution containingmethacrylic acid (10 mM), methacrylamide (20 mM, 1-vinylimidazole (10 mMand riboflavin (1 mM) in 50 mM phosphate buffer pH 7.5, is pumpedthrough the column for 30 min at a flow rate of 0.2 ml/min.Perfluorocyclohexane is pumped through the column at a flow rate of 1ml/min. When all buffer contained in the interstitial pores of thecolumn has been eliminated, the column is illuminated for 2 h under afluorescent tube light source. 10 mM phosphate buffer pH 7.5 is pumpedthrough the column at a flow rate of 1 ml/min and the effluentcollected. The buffer phase containing the polymer is separated from theperfluorocyclohexane phase and freeze-dried. The remainder is dissolvedin 2 ml of 10 mM phosphate buffer pH 7.5, 0.02% Tween-20, andchromatographed on a Sephadex G-5 gel filtration column to remove saltsand unreacted monomers. The eluted polymer fraction is rechromatographedon the IgG-FPLC column, previously equilibrated with 10 mM phosphatebuffer pH 7.5, 0.02% Tween-20. The retained polymer is eluted using astepwise decreasing pH gradient (pH 7.5-3) followed by a solution of 200mM formic acid in water, pH 3, containing 1 M NaCl. The eluted fractionsare freeze-dried, redissolved in water and chromatographed on a SephadexG-5 gel filtration column to remove salts. The ability of the polymerfractions to inhibit lysozyme binding to a sheep-anti-lysozymemonoclonal antibody (the same as used in the moulding step) is evaluatedin a direct ELISA.

[0153] While the invention has been described in terms of preferredembodiments, the skilled artisan -will appreciate that variousmodifications, substitutions, omissions and changes may be made withoutdeparting from the spirit thereof. Accordingly, it is intended that thescope of the present invention be limited solely by the scope of thefollowing claims, including equivalents thereof.

1. A method for producing a compound suitable for in vitro or in vivousage as a diagnostic, therapeutic or prophylactic agent that possessesa complementary structure to a desired molecule or portion thereofcomprising the following steps: (i) selecting a particular molecule towhich a compound having a complementary structure is to be produced;(ii) contacting such molecule with one or more complementary monomersunder conditions wherein such monomers associate around one or moreresidues of such molecule; (iii) effecting polymerization of suchassociated monomers optionally in the presence of at least onecrosslinking agent to produce a polymeric coating that is comprised onthe surface of said molecule and which possesses a complementarystructure to said molecule or a portion thereof; (iv) removing saidmolecule under conditions that result in a polymeric compound thatpossesses a complementary structure to said molecule or portion thereof;and (v) optionally effecting one or more cleavage and/or dissociationsteps to produce compounds that are suitable for in vivo usage as adiagnostic, therapeutic and/or prophylactic agent.
 2. The method ofclaim 1, wherein said molecule is immobilized to a support.
 3. Themethod of claim 1, wherein said molecule is a biomolecule and isselected from the group consisting of a protein, a nucleic acidsequence, a carbohydrate, a peptide, a glycoprotein, a cell, a virus, apathogen, and a tissue.
 4. The method of claim 3, wherein said proteinis selected from the group consisting of an enzyme, antigen, antibody,hormone, receptor, and a fragment thereof.
 5. The method of claim 1,wherein the crosslinking agent comprises at least one cleavablecrosslinker.
 6. The method of claim 5, wherein said crosslinker isselected from the group consisting of bis-acrylcystamine,N,N-diallyltartardiamide, N,N-(1,2-dihydroxyethylen) bisacrylamide, orN,N′-bis-(acryloyl)cystomine,N1-(CE)-1-(4-vinylphenyl)methylidene)-4-vinyl aniline, allyl disulfide,bis(2-(methacyl, 1-oxyethyl)) disulfide.
 7. A method of using thecompound in vivo produced according to claim 1 as a therapeutic ordiagnostic agent comprising administering to a subject in need of suchtreatment a therapeutically or diagnostically effective amount of saidcompound.
 8. The method according to claim 1, wherein the molecule towhich a compound having a complementary structure is to be produced isinsoluble.
 9. The method of claim 8, wherein said molecule is an enzymecrystal or a crosslinked enzyme.
 10. An improved method of affinitypurification which purifies a compound using a compound thatspecifically binds thereto, wherein the improvement comprising using acompound produced according to claim 1 to effect purification.
 11. Animproved assay method which includes the use of a competitive affinityligand wherein the improvement comprises using as the competitiveaffinity ligand a compound produced according to claim
 1. 12. The methodof claim 1, wherein the compound produced by said method is suitable foruse as an active agent selected from the group consisting of a hormone,enzyme, or receptor antagonist or agonist; gene expression modulator,antimicrobial agent, and an anti-tumor agent.
 13. The method of claim 1,wherein the compound which results from said method is subsequentlyattached to a therapeutic or diagnostic agent.
 13. A method forproducing a polymeric compound that exhibits complementary structure toa microbial or mammalian cell comprising the following steps: (i)immobilizing a microbial or mammalian cell to a support; (ii) coatingsaid support and immobilized microbial or mammalian cell with a polymerthat is crosslinkable under specific conditions; (iii) selectivelycrosslinking the portion of the molecular coating that is proximate tothe immobilized mammalian or microbial cell; and (iv) removing theresultant molecular coating from the microbial or mammalian cell. 15.The method of claim 14, wherein the polymer is a photocrosslinkablepolymer.
 16. The method of claim 15, wherein the polymers areas notproximate to the immobilized microbial or mammalian cell are coveredwith a photomask during photocrosslinking.
 17. The method of claim 14,wherein the support is a thin layer support.
 18. The method of claim 14,wherein the polymer coating in step (ii) is introduced by a methodselected from the group consisting of spray-coating, dip-coating, andspin-coating.
 19. The method of claim 14, wherein the resultantpolymeric coating is suitable for use as a cell separating material. 20.The method of claim 14,wherein the molecular coating is subsequentlycleaved into oligomers which function as anti-microbial agents.
 21. Amethod for producing a compound that has a complementary structure tothe active site of a desired molecule comprising the following steps:(i) providing a support which is coated with a first monomer layercoating; (ii) applying to said first monolayer a second layer whichcomprises at least one crosslinkable monomer which is able to movefreely in said second layer; (iii) exposing said second layer to amolecule containing at least one active site and allowing for saidcrosslinkable monomer to associate around said at least one active site;(iv) providing a crosslinking agent and effecting crosslinking toproduce a crosslinked compound that possesses a complementary structureto said at least one active site; and (v) recovering said crosslinkedcompound that possesses a complementary structure to said at least oneactive site or sites.
 22. The method of claim 21, wherein said moleculeis an enzyme or a receptor.
 23. The method of claim 22, wherein theresultant polymeric compound functions as an antagonist or agonist. 24.A method for producing a polymeric or oligomeric compounds that has acomplementary structure to a desired molecule comprises the followingsteps: (i) providing a preformed functionalized polymer that is linearor lightly crosslinked and contacting same with a desired molecularentity such that specific functional groups on the polymer interactcovalently or non-covalently with specific residues on the molecularentity; (ii) allowing for such non-covalent or covalent interactionsbetween the functional groups on the polymer and the molecular entity toequilibrate; (iii) subjecting the resultant equilibrated covalent orcomplex non-covalent between the polymer and the molecular entity be atleast one of the following steps: (1) chemically treating the functionalgroups on the polymer that interact least strongly with the molecularentity by site-selective chemical modification; (2) chemically treatingthe functional groups on the polymer that interact most strongly withthe molecular entity by site-selective chemical modification; and (3)crosslinking the polymer; (iv) separating the molecular entity from theresultant polymer.
 25. The method of claim 24, wherein the molecularentity is in solution or immobilized to a support.
 26. The method ofclaim 24, wherein crosslinking is effected using a reversible cleavablecrosslinking agent.
 27. A compound produced according to claim
 1. 28. Acompound produced according to claim
 14. 29. A compound producedaccording to claim
 21. 30. A compound produced according to claim 25.31. The method of claim 1, wherein the resultant polymer or oligomerranges in molecular weight from about 1000 to about 200,00.
 32. Themethod of claim 31, wherein the molecular weight more preferably rangesfrom about 5,000 to 50,000.
 33. The method of claim 1, wherein the chainlength of the resultant polymer or oligomer ranges from about 25 to 2500angstroms.
 34. The method of claim 33, wherein the chain length morepreferably ranges from 250 to 1000 angstroms.